Reactive oxygen species and free radicals are thought to contribute to many of the adverse effects associated with human diseases and toxicities, but the precise mechanisms through which these toxicities are expressed are not understood adequately. The principle goal of the research described in the present application is to determine these mechanisms. Evidence suggests that loss of homeostatic control of iron, and the oxidations catalyzed by redox-active iron chelates, may be pivotal in tissue damage by reactive oxygen species, but more specific biomarkers and increasingly critical tests of the hypothesis are needed. Other investigators have implicated the loss of protein thiols in mechanisms of oxidant injury, frequently on the basis of exacerbation of injury with 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU), presumably acting through inhibition of glutathione reductase. However, marked shifts in protein thiol/disulfide status are not observed in our studies of reactive oxygen-mediated tissue injury. Cellular compartmentalization or specificity at the molecular level may be significant in thiol/disulfide shifts, and the molecular biological approaches described in the present application provide approaches to testing these hypotheses that also avoid the nonspecific effects of BCNU. A particularly significant observation in our more recent studies has been the dramatic protection against oxidants afforded by selective increases in activities of glutathione reductase in mitochondria, which suggest that the thiol/disulfide status of this compartment is a major determinant of cellular viability. The studies proposed also will test the hypothesis that thiol/disulfide shifts compartmentalized to the mitochondria contribute to oxidant cell injury in vivo, through studies of coenzyme A and its mixed disulfides, and by direct assessments of protein thiols. At doses of acetaminophen below the normal therapeutic doses used in humans, reactive metabolites bind covalently to DNA in mouse liver and kidney in vivo. The binding is not prevented by glutathione, and the recently reported correlation of risk for end stage renal disease with cumulative lifestyle ingestion of acetaminophen suggests a potential relevance of doses of acetaminophen that do not cause necrosis acutely. Understanding the factors responsible for biological exposure to reactive oxygen species, radicals, and other oxidants, the mechanisms through which these reactive intermediates alter biological molecules, and the biochemical and pathophysiological consequences of the respective alterations are important fundamental goals of biochemical research and are the long-term goals of the research described in the present application.